The proposed studies deal with the mechanism by which HIV-1 persists in patients on highly active antiretroviral therapy (HAART). In compliant patients, HAART can decrease levels of free virus in the plasma to below the limit of detection. However, studies completed during the initial funding period demonstrated that HIV-1 persists for life in a small pool of latently infected resting memory CD4 + T cells that carry an integrated copy of the HIV-1 genome. Because latently infected cells represent a major barrier to HIV-1 eradication, it is important to understand the molecular mechanisms that maintain latency. Several potential molecular mechanisms have been proposed based on in vitro studies, but it remains unclear whether any of these accurately account for the persistence of HIV-1 in resting CD4 + T cells in vivo. In the proposed studies, we will use genetic approaches to examine the mechanism of latency in vivo, focusing on the influence of the integration site in the host genome. The first two Specific Aims involve cloning and characterizing sites of HIV-1 integration in resting CD4 + T cells in vivo. A novel method will be used to identify the small subset of integrated proviruses that are functionally significant. Analysis of these integration sites should provide a general picture the chromosomal environments where latent proviruses reside. In the third Specific Aim, we will use integration site information to study the transcriptional activity and chromatin structure of regions of the host chromosome that are sites for HIV-1 integration. In the final Aim, we will test a novel hypothesis concerning the mechanism of HIV-1 latency that is consistent with most published work and that represents a synthesis of several proposed mechanisms. Together, these studies should help to elucidate how HIV-1 latency operates in vivo.